Method of tissue repair using a composite material

ABSTRACT

A composite biocompatible hydrogel material includes a porous polymer matrix, the polymer matrix including a plurality of pores and providing a Young&#39;s modulus of at least 10 GPa. A calcium comprising salt is disposed in at least some of the pores. The porous polymer matrix can comprise cellulose, including bacterial cellulose. The composite can be used as a bone graft material. A method of tissue repair within the body of animals includes the steps of providing a composite biocompatible hydrogel material including a porous polymer matrix, the polymer matrix including a plurality of pores and providing a Young&#39;s modulus of at least 10 GPa, and inserting the hydrogel material into cartilage or bone tissue of an animal, wherein the hydrogel material supports cell colonization in vitro for autologous cell seeding.

CROSS REFERENCE TO RELATED APPLICATION

This application is a divisional of U.S. patent application Ser. No.13/340,114, filed Dec. 29, 2011, which is a divisional of U.S. patentapplication Ser. No. 10/295,461, filed Nov. 15, 2002, now U.S. Pat. No.8,110,222. The entire contents and disclosure of which are incorporatedherein by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

The United States Government has rights in this invention pursuant toContract No. DE-AC05-00OR22725 between the United States Department ofEnergy and UT-Battelle, LLC.

FIELD OF THE INVENTION

The invention relates generally to the fields of biomaterials. Moreparticularly, the invention relates to artificial bone compositions andmethods of forming and using such compositions.

BACKGROUND

Bone grafting is a technique used to repair or help in the healing ofosseous damage caused by procedures and pathologies such as surgery,tumors, trauma, congenital deformities, implant revisions, and jointfusions. The two most common methods presently used to restore bone areallografting and autografting.

Allografting, which comprises transplantation of tissue from a donorinto a host subject, has both clinical and practical drawbacks.Clinically, allografting exposes the host subject to a risk of acquiringan infection and/or other disease such as a host immune system-mediatedanti-graft response. Practical problems with this procedure are thatdonor tissue is often expensive or unavailable.

Autografting, which comprises the transplantation of autologous tissuefrom one site in a subject's body to another, suffers fewer problems ascompared to allografting. For example, because the host and donor arethe same subject, the risk of infection and immune system-mediatedrejection are significantly reduced. However, autografting is stilldisadvantageous in that it requires that two separate surgicalprocedures (one to harvest the tissue; one to transplant the tissue) tobe performed on the subject. Additionally, the supply of usableautologous bone is limited because it is collected from only onesubject.

To avoid problems associated with allografting and autografting,synthetic bone-grafting materials have been developed. Syntheticbone-grafting materials offer numerous clinical and practicaladvantages. The clinical advantages include (1) reduced risk ofinfection and/or rejection and (2) no complications from tissueharvesting surgery. Practical advantages of using synthetic materialsinclude the possible selection of materials that exhibit superiormechanical properties, materials which can be fashioned into custom-madeshapes and sizes, and materials which can be made in large quantities.

A number of different compositions have been used as synthetic materialsfor bone grafting. Predominant among these are calcium phosphates suchas hydroxyapatite. Hydroxyapatite, the main mineral component in bone,is the most stable calcium phosphate form under normal physiologicalconditions. It is a particularly good material for use in bone graftingbecause it readily bonds with bone, and is biocompatible andosteoinductive, permitting bone repair in a location that would notnormally heal if left untreated. Used alone, however, hydroxyapatitelacks mechanical strength and cannot withstand substantial stress.

SUMMARY

A composite biocompatible hydrogel material includes a porous polymermatrix, the polymer matrix including a plurality of pores and providinga Young's modulus of at least 10 GPa. The porous polymer matrix cancomprise cellulose or cellulose derivatives, including bacterialcellulose. A calcium comprising salt is disposed in at least some of thepores.

The calcium salt can include calcium phosphate. Calcium phosphate ispreferably in the form of hydroxyapatite, hydroxyapatite being the moststable form of calcium phosphate and the main mineral component found inbone. The calcium salt may include carbonate or fluoride.

A synthetic bone graft material includes a porous polymer matrix, thepolymer matrix including a plurality of pores and providing a Young'smodulus of at least 10 GPa. A calcium salt is disposed in at least someof the pores. The polymer can include cellulose or cellulosederivatives, such as bacterial cellulose. The calcium salt can includecalcium phosphate in the form of hydroxyapatite. The calcium salt mayinclude carbonate or fluoride. The bone graft material can also includebone morphogenetic proteins (BMP's) or chondrocytes which can bedisposed in at least some of the plurality of pores.

A method of tissue repair within the body of animals includes providinga composite biocompatible hydrogel material including a porous polymermatrix, the polymer matrix including a plurality of pores and providinga Young's modulus of at least 10 GPa, and a calcium comprising saltdisposed in at least some of the pores. The hydrogel material isinserted into cartilage or bone tissue of an animal, wherein thehydrogel material supports cell colonization in vitro for autologouscell seeding.

A method of forming a biocompatible composite hydrogel material includesthe steps of providing a porous polymer matrix, the polymer matrixincluding a plurality of pores and providing a Young's modulus of atleast 10 GPa, and impregnating the pores with a calcium salt. Theimpregnating step can include the steps of immersing the polymer matrixinto a solution containing a calcium source and immersing the polymermatrix in a solution including a source of phosphate, wherein calciumphosphate is formed in the pores. Alternatively, the impregnating stepcan include the steps of phosphorylating the polymer and immersing thephosphorylated polymer in a calcium including solution.

The polymer matrix is preferably generated by bacteria from the genusGluconacetobacter, whereby the polymer formed includes cellulose orcellulose derivatives. The bacteria can be Gluconacetobacter hansenii.The calcium salt can be calcium phosphate, calcium fluoride or calciumcarbonate. Calcium phosphate is preferably in the form ofhydroxyapatite.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is pointed out with particularity in the appended claims.The above and further advantages of this invention may be betterunderstood by referring to the following description taken inconjunction with the accompanying drawings, in which:

FIG. 1 is an X-Ray Diffraction (XRD) pattern of calcium phosphateprecipitated in bacterial cellulose.

FIG. 2 shows a Laser Induced Breakdown Spectroscopy (LIBS) signal forcalcium in bacterial cellulose loaded with calcium phosphate.

FIG. 3 shows a Laser Induced Breakdown Spectroscopy (LIBS) signal forphosphorus in bacterial cellulose loaded with calcium phosphate.

DETAILED DESCRIPTION

A composite biocompatible hydrogel material includes a porous polymermatrix, the polymer matrix including a plurality of pores and providinga Young's modulus of at least 10 GPa, preferably at least 20 GPa, andmore preferably at least 24 GPa. A calcium comprising salt can bedisposed in some of the pores of the polymer. As used herein, the term“hydrogel” refers to a three dimensional networks of hydrophilicpolymers that are insoluble. Since the Young's modulus of human bonegenerally ranges from 12 to 24 GPa, the polymer matrix and resultingcomposite material is strong enough to withstand the stress and strainnecessary for use as a practical orthopedic biomaterial, even in loadbearing applications.

The porosity of the bacterial cellulose polymer enables it to be verywell suited for use as a template for formation of a hydroxyapatitecomposite for bone tissue regeneration. Bacterial cellulose contains99.3% water in its hydrated natural form allowing infusion of both largeand small molecules into its matrix.

The calcium salt can comprise calcium fluoride, calcium carbonate orcalcium phosphate. Calcium phosphate is preferably in the form ofhydroxyapatite, hydroxyapatite being the most stable form of calciumphosphate and the main mineral component of bone. If the composite isused in dental applications, some concentration of fluorine ispreferably included, such as from a calcium salt or calcium fluoride,due to the known cavity preventing properties of fluoride.

Cellulose formed by bacteria is referred to as bacterial cellulose orbiocellulose. This is the preferred form of cellulose for formation ofthe composite material. The Young's modulus of bacterial cellulose hasbeen reported to be 16-18 GPa, and can be improved up to 30 GPa byfurther purification (Yamanaka et al. J. Mat. Sci. 24: 3141-3145 1989).

Bacterial cellulose offers significant advantages over plant cellulosesince it is inherently nearly pure, is highly crystalline, and containsgreater than 99% water. In addition, it forms an intricate highlyinterconnected network of fibers with dimensions of approximately5×0.1×0.004 μm. In contrast, cylindrical cotton fibers are about 70 μm(diam.) and are arranged in parallel bundles. Bacterial cellulose can beobtained commercially from food production companies in Southeast Asiaor synthesized by organisms such as bacteria.

Synthesis by bacteria is known to permit control of the physicalproperties of the cellulose product formed, such as molecular weight andcrystallinity. It may also be possible to directly synthesize cellulosederivatives using bacteria, such as cellulose that contains amine groups(Yamada et al. Biosci. Biotechnol. Biochem. 61: 8 1244-1251 1997).

The preferred species of bacterial for this application are the gramnegative acetic acid bacteria of the genus Acetobacter, reclassified in1997 by Yamada et al. to Gluconoacetobacter xylinus andGluconoacteobacter hansenii (synonyms Gluconacetobacter xylinus andGluconacetobacter hansenii).

G. xylinus and G. hansenii are known to produce a gel-like, hydratedform of cellulose with a highly porous structure. This bacterialcellulose includes a plurality of fibers, the fibers having an averagediameter of about 100 nm.

The composite may be formed by a two step process, described below forformation of a bacterial cellulose calcium phosphate composite. First,samples of bacterial cellulose are permeated by a solution of solublecalcium salt, such as CaCl₂. Other calcium solutions can be used, suchas calcium hydroxide, calcium nitrate, and simulated body fluid.

In a second step, the sample is then soaked in a phosphate salt toprecipitate calcium phosphate in the polymer matrix. Sodium phosphatedibasic is preferred for this purpose. Alternative phosphate solutionsfor the precipitation of calcium phosphate into bacterial celluloseinclude potassium phosphate dibasic, sodium tripolyphosphate, anddiammonium phosphate. In addition, the order of infusion of the saltscan be reversed. This process produces a flexible and durable hydrogelcomposite comprising calcium phosphate crystals deposited within theporous bacterial cellulose matrix. X-ray diffraction studies performedhave confirmed that the calcium phosphate deposited is in the form ofhydroxyapatite, the main mineral component of bone (See FIG. 1). Thepeak at 22 theta corresponds to crystalline cellulose and the otherpeaks are characteristics of calcium deficient hydroxyapatite (PatternDiffraction Database, Mortier et al. J. Solid State Chem. 78: 215 1989).

Optionally, fluoride salts (e.g. from aqueous KF) can be used tofluoridate the hydroxyapatite precipitated in the bacterial cellulose ina separate step. Fluoride is a natural trace element in hydroxyapatiteand is useful in dental applications to prevent cavities. Carbonatesalts (e.g. from aqueous Na₂CO₃) can be used to add carbonate ions ontohydroxyapatite precipitated in the bacterial cellulose to more closelyresemble biological apatite. This may accelerate replacement with bonesince carbonate-apatite can be dissolved significantly faster ascompared to carbonate-free-apatite.

In an alternate method, phosphate groups can be chemically attached tobacterial cellulose using a phosphorylation process. Precipitationoccurs when the phosphorylated cellulose is incubated in calciumcontaining salt solutions. This process can improve the mechanicalproperties of the composite and its ability to retain its shape. Calciumsolutions, such as simulated body fluid, aqueous calcium chloride,aqueous calcium hydroxide, or a combination of the theses solutions canbe used for this purpose.

Unlike pure hydoxyapatite, the composite hydrogel material is neitherstiff nor brittle. It does, however, retain the advantageous property ofhydroxyapatite for bone grafting applications, that being bondable tobone, biocompatible, and osteoinductive. The invention can also be usedfor a wide variety of other biomaterial applications, such as dentalgrafts, ocular implants, or artificial cartilage.

Used as a bone grafting material, bacterial cellulose can be used as acarrier for bone morphogenetic proteins (BMP's). BMP's initiate boneformation and regulate cartilage and hone differentiation in the body.BMP's could be seeded into porous polymers such as native bacterialcellulose or calcium phosphate impregnated bacterial cellulose.

Implanting of chondrocytes into the bacterial cellulose matrix can beused to aid in cartilage reconstruction. Chondrocytes can be seeded intoporous polymers such as native bacterial cellulose or calcium phosphateimpregnated bacterial cellulose.

The composite material is quite suitable for use as a bone implant orrepair material for numerous reasons. First, it is stable when incontact with body fluid and other aqueous solutions. Second, polymerssuch as cellulose have low toxicity and are biocompatible. Third, theporous nature of the polymer selected (e.g. bacterial cellulose) allowsthe composite to support bone ingrowth. Moreover, when bacteriallyproduced polymers are used, the polymers can be made into any shape orsize by growing the bacterial polymers (e.g.) cellulose in appropriatemolds, or sizing the material by cutting.

Other applications for the composite will be apparent to those skilledin the art. For example, the composite hydrogel can also be used forabsorption of materials. Hydroxyapatite immobilized in bacterialcellulose can be used for absorption of proteins. The composite can alsoprovide a hydroxyapatite source for binding water or soil contaminantssuch as Pb, Cd, Zn, U, and Sr or for removal of metal ions from aqueoussolution.

EXAMPLES

The present invention is further illustrated by the following specificexamples. The examples are provided for illustration only and are not tobe construed as limiting the scope or content of the invention in anyway.

Example 1 Deposition of Calcium Phosphate in Bacterial Cellulose

Cubes (1 cm³) of bacterial cellulose obtained from the commercial foodproduct Nata de Coco REM Corporation, Pioneer cor. Sheridan,Mandaluyong, Metro Manila, Philippines were extensively soaked inHPLC-grade distilled water to remove sucrose added during canning. Thecellulose was further cleaned by soaking in 1 M sodium hydroxide,followed by neutralization with acetic acid and soaking in distilledwater to remove salts. The cubes were stored in 20% ethanol. Twelvecellulose cubes were soaked in 50 mM calcium chloride (CaCl₂) (pH 4.83)for 18 hours. A first set of three cubes was then placed in distilledwater as a control. A second set of three cubes was rinsed withdistilled water, then soaked in 100 mM sodium phosphate dibasic(Na₂HPO₄) (pH 9.30). A third set of three cubes was soaked in 100 mMsodium bicarbonate (pH 8.36). And a fourth set of three cubes was soakedin 100 mM sodium carbonate (pH 11.8) for 18 h. All incubations werecarried out at 23° C. After 18 h, the cubes were removed from thesolutions, rinsed with nanopure water, dried, and examined under themicroscope at 1× and at 100× magnification. The bicarbonate andcarbonate samples were observed to contain crystal-like structuresdeposited on the surface of the cellulose. The phosphate sample wascovered with a smooth coating which was rigid and was about three-foldthicker than the control after drying.

In a further experiment, a sample of bacterial cellulose was producedunder controlled laboratory conditions with the strain ATCC 10821Gluconoacetobacter hansenii (obtained from the American Type CultureCollection, Manassas, Va.). A round pellicule of 9 cm diameter and 5 mmthick produced by growth of the bacteria in 150 ml of Schramm-Hestrinmedium (see Example 2) was cleaned with hot water and sodium hydroxide,followed by neutralization with acetic acid, soaking in water, andstorage in 20% ethanol. This pellicule was cut into fourths, and two ofthe four pieces were incubated with 50 mM calcium chloride for 18 h at23° C. with agitation. The calcium solution was decanted and thecellulose pieces were washed with nanopure water. Then 80 ml of sodiumphosphate dibasic, pH 9.3, were added to the cellulose.

After 3 h incubation, the phosphate was decanted and fresh phosphatesolution was added to the cellulose pieces. After another 4 h, thephosphate was decanted, the pieces were washed with nanopure water, andthen the pieces were soaked in 80 ml of calcium chloride for 18 h. Thecalcium solution was then decanted, and the two pieces were again soakedin two 4-h changes of phosphate. Finally, the phosphate was decanted,and the pieces of cellulose were rinsed in nanopure water. One piece ofthe calcium and phosphate cellulose and one piece of untreated controlcellulose were dried on a gel drier and then air-dried. The other pieceswere stored in 20% ethanol to prevent microbial growth. The appearanceand physical properties of the slices of cellulose pellicule treatedwith calcium and phosphate resembled those of the commercial Nata decoco that had been treated with these two salts.

Example 2 Production of Bacterial Cellulose and Modulation of itsProperties

For synthesis of cellulose layers (pellicules), a seed culture of G.hansenii was grown in Schramm-Hestrin medium (Schramm and Hestrin, J.Gen. Microbiol. 11:123, 1954) in a flask under agitated conditions. Thisseed culture is diluted 1/10 with Schramm-Hestrin medium and transferredto sterile containers for static growth. The cellulose is formed at theair-liquid interface. Clouds of cellulose begin to form in the mediumwithin 1-2 days at 23° C. The cellulose pellicules can be harvestedafter 5-8 days depending on the thickness desired. The fastest synthesisof cellulose is seen with glucose as the supplied sugar. Cellulose issynthesized more slowly when the bacteria are grown on medium containingcellobiose, mannitol, or fructose. A synthetic medium (Cannon andAnderson, Crit. Reviews in Microbiol. 17:435, 1991) can be used insteadof the Schramm-Hestrin medium, but cellulose formation is slower than inthe rich medium.

After removal from the surface of the culture dish, the cellulosepellicules were first heated to 90° C. in HPLC-grade distilled water for1-2 h. The pellicules were rinsed with water, then treated with 1%sodium hydroxide for 1-7 days at 23° C. The sodium hydroxide wasneutralized by addition of 1.2 volumes of 500 mM sodium acetate buffer,pH 4.5, followed by soaking in distilled water. The washed pelliculeswere then stored in 20% ethanol. The surface area and shape of thepellicule is determined by the size and shape of the static cultivationcontainer. As the bacteria are aerobic, cellulose production is enhancedwhen the cultures are performed in shallow containers with a totalliquid depth of not more than 5 cm. Containers successfully usedincluded 60- and 100-mm diameter disposable culture dishes, 10 ml glassbeakers, 20×20 cm glass baking dishes, and 2-liter flasks. Growth oflarger pellicules is possible, as shown by the production of cellulosefor Nata de Coco in flat pans of 1×1 m. Utilization of porous containerssuch as polyvinyl chloride gloves for cultivation is known to allowproduction of bacterial cellulose in versatile shapes and sizes.

The physical appearance of the bacterial cellulose was observed to varydependent on the sugar used in the growth medium, the other nutrientsadded to the growth medium, and the agitation of the culture containerduring growth (stationary vs. shaker). When G. hansenii was grown in therich Schramm-Hestrin medium under stationary conditions, a pellicule ofcellulose formed at the air-liquid interface when both glucose andmannitol as the nutrient sugar. When such cultures were incubated inconical flasks on a shaker at low rotational speed, the glucose cultureformed loose clumps, while the mannitol culture form dense, smallpellets and balls. A synthetic medium offers the advantages of lowercost, fewer impurities in the product, and better incorporation ofglucose analogs. Pellicules of bacterial cellulose grown in the richSchramm-Hestrin medium grew fastest and appeared white and dense. Thesepellicules dried to white or translucent membranes about 30-60 micronsthick. Those grown in the synthetic medium grew more slowly and had agel-like, translucent appearance, drying to transparent, very thin,sticky membranes. The pellicules from synthetic medium appear to be moreporous.

Example 3 Identification of Calcium Phosphate

The incorporation of calcium and phosphate in the bacterial cellulosewas quantified using Laser Induced Breakdown Spectroscopy (LIBS) andX-Ray Diffraction (XRD). In LIBS, a laser transforms the material into aplasma spark. The spectral emissions from this spark can be evaluated bya spectrometer to determine the material's elemental breakdown. When anx-ray is directed at a particular substance in the XRD procedure, itwill diffract the x-ray at a specific angle. The unique diffractionpattern can thus be used to identify the material.

Proof of calcium and phosphate induction into the matrix was firstconfirmed using LIBS. The laser used was a Spectra Physics pulsed Nd:YAGlaser (Model INDI-SHG-50) with a fundamental wavelength of 1064 nm. Thefrequency was doubled to give an output wavelength of 532 nm, and thefrequency was quadrupled to give a 266 nm wavelength. The spectrometerwas a 0.5 m Acton spectrometer (Model SP 500) which had a resolution of0.05 nm. The procedure was executed on one sample of bacterial cellulosederived from Gluconacetobacter hansenii in Schramm-Hestrin Media, rinsedin hot water and 2% SDS. The sample was incubated in 200 mL of 50 mMCaCl₂ for 18 hours. It was then rinsed in nanopure water, and soaked in200 mL of 50 mM Na₂HPO₄ for 4 hours. The sample was decanted, added to afresh 200 mL of 50 mM Na₂HPO₄, and incubated for another 4 hours. Afterrinsing in nanopure water, it was again submerged in 200 mL of 50 mMCaCl₂ for 18 hours. It was then rinsed in nanopure water, and soaked in200 mL of 50 mM Na₂HPO₄ for 4 hours. The sample was decanted, added to afresh 200 mL of 50 mM Na₂HPO₄, and incubated for another 4 hours. Allincubations were carried out at 23° C. After a final rinse in nanopurewater, the sample was put on a gel-dryer for 30 minutes.

FIGS. 2 and 3 show LIBS signals for calcium and phosphorus,respectively, in bacterial cellulose loaded with calcium phosphate. Thepeak at 422.8 nm in FIG. 2 indicates the presence of calcium. The peaksat 949 nm and 952.8 nm in FIG. 3 indicate the presence of phosphorus.

An x-ray diffraction analysis of calcium phosphatye precipitated inbacterial cellulose is shown in FIG. 1 As noted earlier, the peak at 22theta corresponds to crystalline cellulose and the other peaks shown arecharacteristics of calcium deficient hydroxyapatite (Ca₉HPO₄(PO₄)₅OH).The measurements were made with a Siemens D5005 X-Ray DiffractionMachine. The sample was run with a step-scanning mode of 20=15.0 and acount time of 20 s. The Cu K tube operated at 40 kV and 20 mA. Thesample used was a 6-cm pellicule derived from Gluconacetobacter hanseniigrown in Schramm-Hestrin media. The cellulose was purified using hotwater and 0.5% NaOH, and 1% SDS. The sample was incubated in 150 mL of100 mM CaCl₂ for 1 day. It was then rinsed with nanopure water, andincubated in 150 mL of 60 mM Na₂HPO₄ for 1 day. This cycle was repeatedtwo more times for a total of 3 daily soakings of 100 mM CaCl₂alternated with 3 daily soakings of 60 mM Na₂HPO₄ carried out at 23° C.It had a final rinse in nanopure water before being put on a gel-dryerfor 30 minutes. The x-ray diffraction pattern matches that ofcalcium-deficient hydroxyapatite (Ca₉HPO₄(PO₄)₅OH) as discovered byMortier et al. (J. of Solid State Chem. 78:215, 1989).

Example 4 Behavior of Bacterial Cellulose in Simulated Body Fluid

When the bacterial cellulose was submerged in simulated body fluid, abiomimetic apatite formed in its matrix. Apatite is identified as anatural, variously colored calcium fluoride phosphate, Ca₅F(PO₄)₃, withchlorine, hydroxyl, or carbonate sometimes replacing the fluoride. Whencalcium phosphate bacterial cellulose was incubated in simulated bodyfluid, its apatite content increased even more significantly. Thisbehavior indicated that bacterial cellulose will act favorably as anorthopedic implant. Instead of being encapsulated by fibrous tissue, theapatite layer induced by the physiological environment material shouldbond directly with bone. Drying and weighing the precipitated samples incomparison to an unaltered sample of bacterial cellulose confirmed thepresence of additional apatite formed when the bacterial cellulose wasexposed to the simulated body fluid. The use of alizarin red S, a dyecommonly used to identify calcium in histology, also indicated thatcalcium was incorporated into the matrix.

Simulated body fluid was prepared from a protocol developed by Tas(Biomaterials 21: 1429-1438 2001). The ion concentrations of thesimulated body fluid mimic that of human plasma (Table 1).

TABLE 1 Ion Concentrations of Simulated Body Fluid compared to HumanPlasma Human Simulated Body Ion Plasma Fluid Na⁺ 142.0 mM  142.0 mM  Cl⁻103.0 mM  125 mM  HCO₃ ⁻ 27.0 mM  27.0 mM  K⁺ 5.0 mM 5.0 mM Mg²⁺ 1.5 mM1.5 mM Ca²⁺ 2.5 mM 2.5 mM HPO₄ ²⁻ 1.0 mM 1.0 mM SO₄ ²⁻ 0.5 mM 0.5 mM

TABLE 2 Chemical Composition of Simulated Body Fluid Solution ReagentSBF (g/L) NaCl 6.547 NaHCO₃ 2.268 KCl 0.373 Na₂HPO₄•2H₂O 5.0 mMMgCl₂•6H₂O 0.305 CaCl₂•2H₂O 0.368The reagents in Table 2 are combined and titrated to pH 7.40 at 37° C. 86-cm bacterial cellulose pellicules were made in an identical fashionfrom the Gluconacetobacter hansenii strain grown in Schramm-Hestrinmedia. The samples were cleaned with hot water and 1% sodium dodecylsulfate/1% sodium hydroxide solution. Two samples were reserved in wateras a control. Four samples were incubated in 200 mL of 100 mM CaCl₂ forone day, followed by incubation in 200 mL of 60 mM Na₂HPO₄ for one day,with 2 more cycles in the same fashion carried out at 23° C. Two of thecalcium phosphate pellicules were incubated in 100 mL of simulated bodyfluid for 11 days at 23° C. The fluid was changed 5 times throughout theduration. Two unaltered samples of cellulose were also incubated insimulated body fluid for 11 days at 23° C., with the solution beingchanged 5 times throughout the duration. The four sets of cellulosesamples were completed as follows: 2 pellicules of unaltered bacterialcellulose, 2 pellicules of bacterial cellulose incubated in simulatedbody fluid, 2 pellicules of calcium-phosphate bacterial cellulose, and 2pellicules of calcium-phosphate bacterial cellulose incubated insimulated body fluid.

One pellicule from each pair was dried on the gel-dryer for 30 minutesand weighed (Table 3).

TABLE 3 Weights of Bacterial Cellulose and Calcium Phosphate BacterialCellulose in Simulated Body Fluid Sample Dry Weight Unaltered BacterialCellulose 0.0428 g Bacterial Cellulose Incubated in 0.0540 g SimulatedBody Fluid Calcium Phosphate Bacterial Cellulose 0.1858 g CalciumPhosphate Bacterial Cellulose 0.2435 g Incubated in Simulated Body FluidThe other pellicules were each stained in 10 mL of 2% Alizarin Red S.The dyed samples were subsequently rinsed in nanopure water and put onthe gel-dryer for 30 minutes. The increase in weight and visualobservation with and without staining indicates apatite formation aftersubmersion in simulated body fluid.

Example 5 Modifying Apatite Formation within the Matrix

The amount of calcium phosphate incorporated into the bacterialcellulose can be altered in at least two ways: (1) varying the number ofsoaking cycles of the calcium chloride and sodium phosphate dibasicsolutions, or (2) by changing the molarity of the calcium chloride andsodium phosphate dibasic solutions.

Varying the Number of Incubation Cycles. Four 10-cm bacterial cellulosepellicules were synthesized from cultures of Gluconacetobacter hanseniistrain grown in Schramm-Hestrin media as described above. The pelliculeswere cleaned with hot water and 2% sodium dodecyl sulfate. One pelliculewas stored in water as a control. A second pellicule was incubated in150 mL of 50 mM CaCl₂ for 18 hours, and then rinsed in nanopure water.The cellulose was then incubated in 150 mL of 100 mM Na₂HPO₄ for 4hours, decanted, and then added to another 150 mL of 100 mM Na₂HPO₄ foranother 4 hours. The cellulose was rinsed, and again suspended in 150 mLof 50 mM CaCl₂ for 18 hours, then rinsed in nanopure water. Thecellulose was again incubated in 150 mL of 100 mM Na₂HPO₄ for 4 hours,decanted, and then added to another 150 mL of 100 mM Na₂HPO₄ for another4 hours. The pellicule went through another soaking in CaCl₂ and Na₂HPO₄for a total of 3 cycles of incubation. The third pellicule underwent 4cycles of CaCl₂ incubation followed by Na₂HPO₄ incubation. The lastpellicule undertook 5 cycles of CaCl₂ incubation followed by a Na₂HPO₄incubation. All incubations were carried out at 23° C. The 4 pelliculeswere weighed in their hydrated state. The samples were then put on agel-dryer for 30 minutes, and baked in a 90° C. oven until a constantweight was reached (Table 4).

TABLE 4 Weights of Bacterial Cellulose Precipitated in Varying Number ofSolution Cycles Wet Dry Sample Weight Weight Unaltered BacterialCellulose 1.00 g 0.0690 g Bacterial Cellulose Incubated in 3 2.73 g0.3186 g Cycles of 50 mM CaCl₂ and Sodium Phosphate Dibasic SolutionsBacterial Cellulose Incubated in 4 cycles 3.40 g 0.5940 g of CalciumChloride and Sodium Phosphate Dibasic Solutions Bacterial CelluloseIncubated in 5 cycles 4.55 g 0.6798 g of Calcium Chloride and SodiumPhosphate Dibasic SolutionsThe above results show an incremental weight gain with the increasingnumber of cycles of incubation in the 2 solutions. This indicates thatthe amount of calcium phosphate grows with the number of solutioncycles.

The effect of varying the molarity of the immersion solutions wasinvestigated. Ten 9-cm bacterial cellulose pellicules were synthesizedfrom cultures of Gluconacetobacter hansenii strain grown inSchramm-Hestrin media as described above. The samples were cleaned withhot water and 2% sodium dodecyl sulfate. Two of the pellicules werestored in water as controls. Each of the 8 pellicules were incubated ina particular molarity of CaCl₂ solution for 1 day, followed byincubation in a particular molarity of Na₂PO₄ for 1 day, then incubatedagain for 1 day in the aqueous CaCl₂, and again in a last soaking of theaqueous Na₂PO₄ for 1 day. All suspensions were carried out at 23° C. Themolarity ratio of Ca to P was kept to 10:6 in each sample to mimic theCa:P concentration of hydroxyapatite. The concentrations of calciumchloride ranged from 25 mM to 200 mM, and the sodium phosphate dibasicconcentrations varied from 15 mM to 120 mM (Table 5).

TABLE 5 Weights of Bacterial Cellulose Incubated in VaryingConcentrations Of Calcium Chloride and Sodium Phosphate Dibasic Molarityof Molarity of CaCl₂ used in Na₂HPO₄ Used in Wet Dry Sample IncubationIncubation Weight Weight 1 200 mM 120 mM  12.49 g  0.7865 g 2 175 mM 105mM  11.40 g  0.6682 g 3 150 mM 90 mM 11.13 g  0.6015 g 4 125 mM 75 mM8.70 g 0.5065 g 5 100 mM 60 mM 7.56 g 0.2948 g 6  75 mM 45 mM 6.91 g0.3104 g 7  50 mM 30 mM 8.49 g 0.3757 g 8  25 mM 15 mM 8.12 g 0.1940 g 9No Incubation No Incubation 13.79 g  0.1028 gUpon completion of the last cycle, the pellicules were rinsed innanopure water and weighed. The samples were put on a gel-dryer for 30minutes, and weighed again. Table 5 displays the wet and dry weights foreach sample. Although the data was inconsistent with samples 6 and 7, itis evident that increasing molarity concentrations of the soakingsolutions causes increased weight gain in the pellicules. This indicatesthe increased synthesis of calcium phosphate.

Example 6 Alternative Reagents

Salt solutions other than calcium chloride and sodium phosphate dibasiccan be used to precipitate calcium phosphate into bacterial cellulose.For example, potassium phosphate dibasic and sodium tripolyphosphate canbe employed as the phosphate donor. Concentrated calcium hydroxide canbe used as a calcium donor.

Potassium phosphate dibasic was used as a phosphate donor in anexperiment. 3-6 cm pellicules derived from Gluconacetobacter hanseniiand Schramm-Hestrin media were cleaned in hot water, 1% SDS/0.5% NaOHsolution. One pellicule was reserved as control. The second pelliculewas suspended in 100 mL of 100 mM CaCl₂ for 1 day, and then added to 100mL of 60 mM K₂HPO₄. Precipitation was immediately observed. Thecellulose went through 2 more additional daily cycles of 100 mM CaCl₂alternated with 2 daily cycles of 60 mM K₂HPO₄. The precipitated sampleis significantly whiter, stiffer, and thicker than the control. A thirdpellicule was precipitated by the original protocol using 100 mM CaCl₂and 60 mM Na₂HPO₄. It was immersed in 3 daily cycles of the calciumchloride alternated with 3 daily cycles of the sodium phosphate dibasic.All three samples were weighed in their hydrated state. Then they wereput on a gel-dryer for 30 minutes and re-weighed (Table 6).

TABLE 6 Weight of Ca—P Precipitated Bacterial Cellulose Using DifferentPO₄ ⁻ Donors Wet Dry Sample Weight Weight Bacterial Cellulose Immersedin CaCl_(2 (aq)) 7.576 g 0.3595 g and K₂HPO_(4 (aq)) Bacterial CelluloseImmersed in CaCl_(2 (aq)) 7.678 g 0.2996 and Na₂HPO_(4 (aq)) UnalteredBacterial Cellulose 8.937 g 0.0400 gThe similar results of potassium phosphate dibasic as compared to thesodium phosphate dibasic confirm that it can be used as alternativephosphate solution.

Sodium tripolyphosphate was used as a phosphate donor in an experiment.Sodium Tripolyphosphate (Na₅P₃O₁₀) (STPP) has a peculiar behavior in theprecipitation of calcium phosphate in the cellulose matrix. It caneither have the role of a phosphate donor, or as an apatite eliminator.STPP is a water softener which will substitute sodium ions for calciumions. However, when the cellulose was soaked in calcium chloride, thenadded to STPP solution, and again soaked in CaCl₂; a heavy calciumphosphate precipitate formed. When a pellicule that has alreadyincorporated calcium phosphate into its matrix was soaked in STPP, thesodium ions of the STPP substituted the calcium ions in the precipitatethereby removing almost all of the apatite.

Two 6-cm pellicules derived from a culture of Gluconacetobacter hanseniiin Schramm-Hestrin media were cleaned in hot water, 1% SDS/0.5% NaOHsolution. Each pellicule was soaked in 3 daily cycles of 100 mL of 100mM CaCl₂ alternated with 3 daily cycles of 100 mL of 60 mM Na₅P₃O₁₀ for1 day. One pellicule was removed after the last cycle of STPP. The otherwas soaked in an additional treatment of 100 mL of 100 mM CaCl₂ for oneday. The pellicule which had its last soaking in STPP was practicallydevoid of apatite. It appeared similar to an unaltered sample ofbacterial cellulose. The pellicule that had its last incubation in CaCl₂was richly deposited with calcium phosphate. It is much whiter andthicker than the former pellicule.

Calcium hydroxide can be used as a calcium donor. Three-6 cm pelliculesderived from a culture of Gluconacetobacter hansenii in Schramm-Hestrinmedia were cleaned in hot water, 1% SDS/1% NaOH solution. One pelliculewas reserved as a control. Two pellicules were suspended in 100 mL of 12mM Ca(OH)₂ for 1 day, and then added to 100 mL of 60 mM Na₂HPO₄.Precipitation was immediately observed. The cellulose went through 2additional daily cycles of 12 mM Ca(OH)₂ alternated with 2 daily cyclesof 60 mM Na₂HPO₄. The precipitated sample was significantly whiter,stiffer, and thicker than the control. One of the precipitated sampleswas dried and weighed while the other was stained with alizarin red Sfor calcium detection. The weight of the precipitated pellicule was0.110 g while the unaltered pellicule was 0.0329 g.

Example 7 Apatite Formation in Phosphorylated Cellulose

Phosphate groups were chemically attached to the bacterial celluloseusing a phosphorylation process. Precipitation occurred when thephosphorylated cellulose was incubated in salt solutions. This improvedthe mechanical properties of the composite and its ability to retain itsshape.

Five 6-cm pellicules derived from a culture of Gluconacetobacterhansenii in Schramm-Hestrin media were cleaned in a hot water, 1%SDS/0.5% NaOH solution. The volume of the pellicules was approximately38.7 mL, derived from weight. The pellicules were incubated for one dayin 45% urea, and incubated for another day in a fresh solution of 45%urea, making their concentration about 41% urea. The pellicules werethen incubated in a 42% phosphoric acid/75% urea solution until thepellicules had an approximate concentration of 31% phosphoric acid/53%urea. They were then put in a 120° C. oven for 3 hours, and then rinsedin nanopure water.

One of the phosphorylated pellicules was incubated in 100 mM CaCl₂ for 2weeks (solution was changed 8 times). Precipitation was evident. Thecellulose became whiter, denser, and more robust after incubation. Thesample was put on a gel-dryer for 30 minutes. Its weight was found to be0.0292 g. A control sample of phosphorylated cellulose that was notincubated was also dried for comparison. Due to its poor mechanicalproperties and thin composition, however, it was unable to be fullyrecovered from the gel-dryer. This further proves the strengtheningbehavior of incubating the phosphorylated cellulose in salt solutions.

A sample of phosphorylated bacterial cellulose prepared in theabove-described procedure was incubated in simulated body fluid for 12days. The solution was changed 7 times. After treatment, the cellulosewas more robust, durable, and had a slight white precipitate. The sampleis put on a gel-dryer for 30 minutes. Its weight was found to be 0.0309g.

A sample of phosphorylated bacterial cellulose was incubated insaturated calcium hydroxide (about 12 mM) for 9 days (solution waschanged 5 times). As with the above-described solutions, the cellulosebecame more robust and durable. But unlike the above-describedsolutions, calcium hydroxide produced significantly more whiteprecipitate in the matrix. The sample was put on a gel-dryer for 30minutes. Its weight was found to be 0.0166 g.

Soaking of the phosphorylated bacterial cellulose in this combination ofsolutions produced the most precipitate. A sample of phosphorylatedbacterial cellulose was incubated in saturated calcium hydroxide (about12 mM) for 9 days (solution was changed 5 times). Subsequently, thesample was incubated in simulated body fluid for 9 days (the solutionwas changed 5 times). The precipitate was more abundant and whiter thanthe sample incubated in calcium hydroxide alone. The sample was put on agel-dryer for 30 minutes. Parching the sample caused it to become muchmore brittle and white with apatite. The weight was found to be 0.0772g.

Example 8 Incorporation of Other Calcium Salts

Additional immersion solutions were investigated to see if other calciumsalts, such as calcium fluoride and calcium carbonate, could beintegrated into bacterial cellulose. Because physiologicalhydroxyapatite possesses strains of fluoride and carbonate, using thesesolutions may be utilized to produce a more biomimetic ceramic. Fluorideis known to be beneficial to dental implants due to its ability toinhibit cavities.

A 6-cm bacterial cellulose pellicule derived from Gluconacetobacterhansenii grown in Schramm-Hestrin media was cleaned in a hot water, 1%SDS/0.5% NaOH solution. The pellicule was soaked in 100 mL of 100 mMCaCl₂ for one day, then soaked in 100 mL of 200 mM KF. Precipitation wasimmediately observed. The pellicule was incubated for 2 more dailycycles of CaCl₂ alternated with 2 daily cycles of KF. The cellulosebecame very white and thick with precipitate. After drying, its weightwas measured to be 0.2496 g. The identification of the salt is suspectedto be calcium fluoride.

In another experiment, two 10-cm bacterial cellulose pellicules fromGluconacetobacter hansenii grown on synthetic glucose media were cleanedwith hot water and 1% sodium hydroxide. The pellicules were soaked in100 mL of 100 mM CaCl₂ for one day, then soaked in 100 mL of 100 mMNa₂CO₃. Precipitation was immediately observed. The pellicules wereincubated for 2 more daily cycles of CaCl₂ alternated with 2 dailycycles of Na₂CO₃. When placed into acetic acid, an effervescent reactionwas observed indicating that the salt in the matrix might be calciumcarbonate.

Other Embodiments

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

What is claimed is:
 1. A method for producing a biocompatible hydrogelmaterial, the process comprising providing bacterial cellulose whichincludes a plurality of pores, immersing said bacterial cellulose in asolution containing a calcium source, and immersing said bacterialcellulose in a separate solution containing a source of phosphate toform said hydrogel material.
 2. The method of claim 1, wherein saidcalcium source is at least one selected from the group consisting ofcalcium phosphate, calcium chloride, calcium fluoride, calciumcarbonate, calcium hydroxide, and calcium nitrate.
 3. The method ofclaim 1, wherein said source of phosphate is at least one selected fromthe group consisting of sodium phosphate dibasic, potassium phosphatedibasic, sodium tripolyphosphate, and diammonium phosphate.
 4. Themethod of claim 1, wherein the immersing in said solution containing acalcium source is performed before the immersing in said solutioncontaining a source of phosphate.
 5. The method of claim 1, wherein theimmersing in said solution containing a calcium source is performedafter the immersing in said solution containing a source of phosphate.6. The method of claim 1, wherein the immersing in said solutioncontaining a calcium source and the immersing in said solutioncontaining a source of phosphate are performed in multiple cycles. 7.The method of claim 5, wherein said solution containing a source ofphosphate is a solution containing phosphoric acid and urea, and thebacterial cellulose becomes phosphorylated after immersing in saidsolution containing a source of phosphate.